The Anti-apoptotic Effect of Ghrelin on Restraint Stress-Induced Thymus Atrophy in Mice.

Thymic atrophy is a complication that results from exposure to many environmental stressors, disease treatments, and microbial challenges. Such acute stress-associated thymic loss can have a dramatic impact on the host's ability to replenish the necessary naïve T cell output to reconstitute the peripheral T cell numbers and repertoire to respond to new antigenic challenges. We have previously reported that treatment with the orexigenic hormone ghrelin results in an increase in the number and proliferation of thymocytes after dexamethasone challenge, suggesting a role for ghrelin in restraint stress-induced thymic involution and cell apoptosis and its potential use as a thymostimulatory agent. In an effort to understand how ghrelin suppresses thymic T cell apoptosis, we have examined the various signaling pathways induced by receptor-specific ghrelin stimulation using a restraint stress mouse model. In this model, stress-induced apoptosis in thymocytes was effectively blocked by ghrelin. Western blot analysis demonstrated that ghrelin prevents the cleavage of pro-apoptotic proteins such as Bim, Caspase-3, and PARP. In addition, ghrelin stimulation activates the Akt and Mitogen-activated protein kinases (MAPK) signaling pathways in a time/dose-dependent manner. Moreover, we also revealed the involvement of the FoxO3a pathway in the phosphorylation of Akt and ERK1/2. Together, these findings suggest that ghrelin inhibits apoptosis by modulating the stress-induced apoptotic signal pathway in the restraint-induced thymic apoptosis.

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways.

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levels and impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo.

Tellurium compound AS101 ameliorates experimental autoimmune encephalomyelitis by VLA-4 inhibition and suppression of monocyte and T cell infiltration into the CNS.

Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system (CNS) involving demyelinating and neurodegenerative processes. Several of the major pathological CNS alterations and behavioral deficits of MS are recapitulated in the experimental autoimmune encephalitis (EAE) mouse model in which the disease process is induced by administration of myelin peptides. Development of EAE requires infiltration of inflammatory cytokine-generating monocytes and macrophages, and auto-reactive T cells, into the CNS. Very late antigen-4 (VLA-4, α4ß1) is an integrin molecule that plays a role in inflammatory responses by facilitating the migration of leukocytes across the blood-brain barrier during inflammatory disease, and antibodies against VLA-4 exhibit therapeutic efficacy in mouse and monkey MS models. Here, we report that the tellurium compound AS101 (ammonium trichloro (dioxoethylene-o,o') tellurate) ameliorates EAE by inhibiting monocyte and T cell infiltration into the CNS. CD49d is an alpha subunit of the VLA-4 (α4ß1) integrin. During the peak stage of EAE, AS101 treatment effectively ameliorated the disease process by reducing the number of CD49d(+) inflammatory monocyte/macrophage cells in the spinal cord. AS101 treatment markedly reduced the pro-inflammatory cytokine levels, while increasing anti-inflammatory cytokine levels. In contrast, AS101 treatment did not affect the peripheral populations of CD11b(+) monocytes and macrophages. AS101 treatment reduced the infiltration of CD4(+) and CD49(+)/VLA4 T cells. In addition, treatment of T cells from MS patients with AS101 resulted in apoptosis, while such treatment did not affect T cells from healthy donors. These results suggest that AS101 reduces accumulation of leukocytes in the CNS by inhibiting the activity of the VLA-4 integrin and provide a rationale for the potential use of Tellurium IV compounds for the treatment of MS.

Arctigenin suppresses receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast differentiation in bone marrow-derived macrophages.

Osteoclasts, multinucleated bone-resorbing cells, are closely associated with bone diseases such as rheumatoid arthritis and osteoporosis. Osteoclasts are derived from hematopoietic precursor cells, and their differentiation is mediated by two cytokines, including macrophage colony stimulating factor and receptor activator of nuclear factor κB ligand (RANKL). Previous studies have shown that arctigenin exhibits an anti-inflammatory effect. However, the effect of arctigenin on osteoclast differentiation is yet to be elucidated. In this study, we found that arctigenin inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages in a dose-dependent manner and suppressed RANKL-mediated bone resorption. Additionally, the expression of typical marker proteins, such as NFATc1, c-Fos, TRAF6, c-Src, and cathepsin K, were significantly inhibited. Arctigenin inhibited the phosphorylation of Erk1/2, but not p38 and JNK, in a dose-dependent manner. Arctigenin also dramatically suppressed immunoreceptor tyrosine-based activation motif-mediated costimulatory signaling molecules, including Syk and PLCγ2, and Gab2. Notably, arctigenin inhibited the activation of Syk through RANKL stimulation. Furthermore, arctigenin prevented osteoclast differentiation in the calvarial bone of mice following stimulation with lipopolysaccharide. Our results show that arctigenin inhibits osteoclast differentiation in vitro and in vivo. Therefore, arctigenin may be useful for treating rheumatoid arthritis and osteoporosis.

The GHS-R blocker D-[Lys3] GHRP-6 serves as CCR5 chemokine receptor antagonist.

[D-Lys3]-Growth Hormone Releasing Peptide-6 (DLS) is widely utilized in vivo and in vitro as a selective ghrelin receptor (GHS-R) antagonist. This antagonist is one of the most common antagonists utilized in vivo to block GHS-R function and activity. Here, we found that DLS also has the ability to modestly block chemokine function and ligand binding to the chemokine receptor CCR5. The DLS effects on RANTES binding and Erk signaling as well as calcium mobilization appears to be much stronger than its effects on MIP-1α and MIP-1ß. CCR5 have been shown to act as major co-receptor for HIV-1 entry into the CD4 positive host cells. To this end, we also found that DLS blocks M-tropic HIV-1 propagation in activated human PBMCs. These data demonstrate that DLS may not be a highly selective GHS-R1a inhibitor and may also effects on other G-protein coupled receptor (GPCR) family members. Moreover, DLS may have some potential clinical applications in blocking HIV infectivity and CCR5-mediated migration and function in various inflammatory disease states.

Identification of ghrelin receptor blocker, D-[Lys3] GHRP-6 as a CXCR4 receptor antagonist.

[D-Lys3]-Growth Hormone Releasing Peptide-6 (DLS) is widely utilized in vivo and in vitro as a selective ghrelin receptor (GHS-R) antagonist. Unexpectedly, we identified that DLS also has the ability to block CXCL12 binding and activity through CXCR4 on T cells and peripheral blood mononuclear cells (PBMCs). Moreover, as CXCR4 has been shown to act as a major co-receptor for HIV-1 entry into CD4 positive host cells, we have also found that DLS partially blocks CXCR4-mediated HIV-1 entry and propagation in activated human PBMCs. These data demonstrate that DLS is not the specific and selective antagonist as thought for GHS-R1a and appears to have additional effects on the CXCR4 chemokine receptor. Our findings also suggest that structural analogues that mimic DLS binding properties may also have properties of blocking HIV infectivity, CXCR4 dependent cancer cell migration and attenuating chemokine-mediated immune cell trafficking in inflammatory disorders.

The Src family kinase Fgr is critical for activation of mast cells and IgE-mediated anaphylaxis in mice.

Mast cells are critical for various allergic disorders. Mast cells express Src family kinases, which relay positive and negative regulatory signals by Ag. Lyn, for example, initiates activating signaling events, but it also induces inhibitory signals. Fyn and Hck are reported to be positive regulators, but little is known about the roles of other Src kinases, including Fgr, in mast cells. In this study, we define the role of Fgr. Endogenous Fgr associates with FcεRI and promotes phosphorylation of Syk, Syk substrates, which include linkers for activation of T cells, SLP76, and Gab2, and downstream targets such as Akt and the MAPKs in Ag-stimulated mast cells. As a consequence, Fgr positively regulates degranulation, production of eicosanoids, and cytokines. Fgr and Fyn appeared to act in concert, as phosphorylation of Syk and degranulation are enhanced by overexpression of Fgr and further augmented by overexpression of Fyn but are suppressed by overexpression of Lyn. Moreover, knockdown of Fgr by small interfering RNAs (siRNAs) further suppressed degranulation in Fyn-deficient bone marrow-derived mast cells. Overexpression of Fyn or Fgr restored phosphorylation of Syk and partially restored degranulation in Fyn-deficient cells. Additionally, knockdown of Fgr by siRNAs inhibited association of Syk with FcεRIγ as well as the tyrosine phosphorylation of FcεRIγ. Of note, the injection of Fgr siRNAs diminished the protein level of Fgr in mice and simultaneously inhibited IgE-mediated anaphylaxis. In conclusion, Fgr positively regulates mast cell through activation of Syk. These findings help clarify the interplay among Src family kinases and identify Fgr as a potential therapeutic target for allergic diseases.

A novel imidazo[1,5-b]isoquinolinone derivative, U63A05, inhibits Syk activation in mast cells to suppress IgE-mediated anaphylaxis in mice.

Mast cells play a pivotal role in IgE-mediated allergic responses. Development of specific inhibitors against FcεRI-associated proximal signaling molecules in mast cells may represent a promising therapeutic strategy for allergic diseases. We examined whether a novel synthetic compound, 3-butyl-1-chloro-8-(2-methoxycarbonyl)phenyl-5H-imidazo[1,5-b]isoquinolin-10-one (U63A05), could suppress antigen-stimulated degranulation and cytokine secretion in mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. U63A05 reversibly and dose-dependently inhibited degranulation of rat basophilic leukemia (RBL)-2H3 mast cells and bone marrow-derived mast cells (BMMCs) stimulated by antigen (IC(50) values for RBL-2H3 and BMMCs were 4.1 and 4.8 µM, respectively). The secretion of inflammatory cytokines was also suppressed in antigen-stimulated mast cells. However, degranulation by thapsigargin, a typical calcium inducer, was not inhibited by U63A05. U63A05 exerts its inhibitory effect, to the same extent as in degranulation, on the activating phosphorylation of Syk and downstream signaling molecules, including LAT and SLP-76. Further downstream, the activating phosphorylations of Akt, Erk1/2, p38, and JNK were also inhibited. Finally, antigen-stimulated PCA was dose-dependently suppressed in mice (ED(50), 26.3 mg/kg). Taken together, the results suggest that U63A05 suppresses the activation of mast cells and the mast cell-mediated allergic response through the inhibition of Syk activation in mast cells.

Interleukin-32α production is regulated by MyD88-dependent and independent pathways in IL-1ß-stimulated human alveolar epithelial cells.

Interleukin (IL)-32 is a recently described cytokine that appears to play a critical role in a variety of inflammatory diseases including chronic obstructive pulmonary disease (COPD). However, thus far, the regulation of IL-32 production has not been fully established. Here, we report on signaling pathways that regulate the production of IL-32α, the most abundant isoform, in the human alveolar epithelial cell line, A549. IL-32α was expressed and secreted by IL-1ß. The IL-32 expression was attenuated by PP2 (a Src-family kinase [SFK] inhibitor), rottlerin (a protein kinase [PK] Cδ inhibitor), and LY294002 (a phosphatidylinositol 3-kinase [PI3K] inhibitor). Furthermore, the overexpression of Fgr rather than other SFKs upregulated IL-32α expression, while Fgr small interfering RNA (siRNA) transfection downregulated it. The suppression of Fgr with PP2 and Fgr siRNA inhibited activating phosphorylation of PKCδ and PI3K/Akt, but not IL-1 receptor-associated kinase (IRAK)1, a well-known MyD88-dependent signaling molecule, and Erk1/2, p38, and JNK. Rottlerin and PKCδ siRNA also inhibited expression of IL-32α and activation of PI3K/Akt, but not of IRAK1 and mitogen activation protein (MAP) kinases. MyD88 siRNA suppressed the expression of IL-32α and the phosphorylation of IRAK1, PI3K, and MAP kinases, but not of PKCδ. Of interest, both Fgr/PKCδ and MyD88-dependent signals regulated PI3K/Akt, suggesting that it is a crosstalk molecule. Among MyD88-dependent MAP kinases, only p38 regulated IL-32α expression and PI3K/Akt activation. With these results, we demonstrated that the expression and secretion of IL-32α are regulated by MyD88-dependent IRAK1/p38/PI3K and independent Fgr/PKCδ/PI3K pathways, and that Fgr and PKCδ are critical for the MyD88-independent IL-32α production.

Interleukin-33 stimulates formation of functional osteoclasts from human CD14(+) monocytes.

Interleukin (IL)-33 is a recently described pro-inflammatory cytokine. Here we demonstrate IL-33 as a regulator of functional osteoclasts (OCs) from human CD14(+) monocytes. IL-33 stimulates formation of tartrate-resistant acid phosphatase (TRAP)(+) multinuclear OCs from monocytes. This action was suppressed by anti-ST2 antibody, suggesting that IL-33 acts through its receptor ST2, but not by the receptor activator of NF-κB ligand (RANKL) decoy, osteoprotegerin, or anti-RANKL antibody. IL-33 stimulated activating phosphorylations of signaling molecules in monocytes that are critical for OC development. These included Syk, phospholipase Cγ2, Gab2, MAP kinases, TAK-1, and NF-κB. IL-33 also enhanced expression of OC differentiation factors including TNF-α receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells cytoplasmic 1, c-Fos, c-Src, cathepsin K, and calcitonin receptor. IL-33 eventually induced bone resorption. This study suggests that the osteoclastogenic property of IL-33 is mediated through TRAF6 as well as the immunoreceptor tyrosine-based activation motif-dependent Syk/PLCγ pathway in human CD14(+) monocytes.

Oral administration of curcumin suppresses production of matrix metalloproteinase (MMP)-1 and MMP-3 to ameliorate collagen-induced arthritis: inhibition of the PKCdelta/JNK/c-Jun pathway.

We investigated whether oral administration of curcumin suppressed type II collagen-induced arthritis (CIA) in mice and its effect and mechanism on matrix metalloproteinase (MMP)-1 and MMP-3 production in CIA mice, RA fibroblast-like synoviocytes (FLS), and chondrocytes. CIA in mice was suppressed by oral administration of curcumin in a dose-dependent manner. Macroscopic observations were confirmed by histological examinations. Histological changes including infiltration of immune cells, synovial hyperplasia, cartilage destruction, and bone erosion in the hind paw sections were extensively suppressed by curcumin. The histological scores were consistent with clinical arthritis indexes. Production of MMP-1 and MMP-3 were inhibited by curcumin in CIA hind paw sections and tumor necrosis factor (TNF)-alpha-stimulated FLS and chondrocytes in a dose-dependent manner. As for the mechanism, curcumin inhibited activating phosphorylation of protein kinase Cdelta (PKCdelta) in CIA, FLS, and chondrocytes. Curcumin also suppressed the JNK and c-Jun activation in those cells. This study suggests that the suppression of MMP-1 and MMP-3 production by curcumin in CIA is mediated through the inhibition of PKCdelta and the JNK/c-Jun signaling pathway.

Morin inhibits Fyn kinase in mast cells and IgE-mediated type I hypersensitivity response in vivo.

Mast cells are responsible for IgE-mediated allergic responses. Although dietary flavonoid morin has been known to suppress mast cell activation, its in vivo anti-allergic activity and the underlying mechanisms remain are largely unknown. In this study, we determine whether morin suppresses IgE-mediated allergic responses in an animal model and its mechanism of action. Morin suppressed IgE-mediated PCA in mice (ED50 23.9 mg/kg) and inhibited degranulation and production of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-4 in antigen (Ag)-stimulated mast cells. The mechanism of action was a follows. Morin inhibited the activating phosphorylation of spleen tyrosine kinase (Syk) and linker for activation of T cells (LAT) in rat basophilic leukemia (RBL)-2H3 cells and bone marrow-derived mast cells (BMMCs). Akt and the mitogen-activated protein (MAP) kinases, p38, extracellular signal-regulated kinase (ERK)1/2, and c-Jun N-terminal kinase (JNK) were inhibited as well. In vitro kinase assay indicated that Fyn kinase, not Lyn and Syk, was inhibited by morin in a dose-dependent manner (IC50 5.7 microM). In conclusion, the results suggest that morin suppresses the IgE-mediated allergic response by primarily inhibiting Fyn kinase in mast cells.

Tumor necrosis factor alpha-induced interleukin-32 is positively regulated via the Syk/protein kinase Cdelta/JNK pathway in rheumatoid synovial fibroblasts.

OBJECTIVE: Interleukin-32 (IL-32) is a recently discovered cytokine that appears to play a critical role in human rheumatoid arthritis (RA). It is highly expressed in synovium and fibroblast-like synoviocytes (FLS) from RA patients, but not in patients with osteoarthritis (OA). This study was undertaken to assess IL-32 levels in RA synovial fluid (SF) and to investigate the secretion and regulation of IL-32 in RA FLS. METHODS: FLS and SF were obtained from the joints of RA patients. The secretion and expression of IL-32 and activation of signaling molecules were examined by enzyme-linked immunosorbent assay, immunoblotting, immunoprecipitation, reverse transcriptase-polymerase chain reaction, and small interfering RNA (siRNA) transfection. RESULTS: IL-32 levels were high in RA SF compared with OA SF. Furthermore, RA FLS expressed and secreted IL-32 when stimulated with tumor necrosis factor alpha (TNFalpha). TNFalpha-induced expression of IL-32 was significantly suppressed, in a dose-dependent manner, by inhibitors of Syk, protein kinase Cdelta (PKCdelta), and JNK and by knockdown of these kinases and c-Jun with siRNA. We also observed that PKCdelta mediated the activation of JNK and c-Jun, and experiments using specific inhibitors and siRNA demonstrated that Syk was the upstream kinase for the activation of PKCdelta. CONCLUSION: The present findings suggest that IL-32 may be a newly identified prognostic biomarker in RA, thereby adding valuable knowledge to the understanding of this disease. The results also demonstrate that the production of IL-32 in RA FLS is regulated by Syk/PKCdelta-mediated signaling events.

Mast cell-mediated allergic response is suppressed by Sophorae flos: inhibition of SRC-family kinase.

Complementary and alternative medicines are considered as a promising direction for the development of anti-allergic therapies in oriental countries. We screened approximately 100 oriental herbal medicines for anti-allergic activity. Sophorae flos exhibited the most potent effect on degranulation in antigen-stimulated mast cells. We further investigated the effect of Sophorae flos on the IgE-mediated allergic response in vivo and its mechanism of action in mast cells. Sophorae flos exhibited a significant inhibitory effect on degranulation in antigen-stimulated mast cells with IC(50) values of approximately 31.6 microg/mL (RBL-2H3 mast cells) and approximately 47.8 microg/mL (bone marrow-derived mast cells). Sophorae flos also suppressed the expression and secretion of TNF-alpha and IL-4 in the cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Sophorae flos inhibited the activating phosphorylation of Syk and LAT in mast cells. Further downstream, activating phosphorylation of Akt and the prototypic MAP kinases, namely, p38, ERK1/2, and JNK, were also inhibited. These results suggest that Sophorae flos inhibits the Src family kinase-dependent signaling cascades in mast cells and may thus exert anti-allergic activity.

Curcumin, a constituent of curry, suppresses IgE-mediated allergic response and mast cell activation at the level of Syk.

BACKGROUND: Activation of mast cells through the high-affinity receptor for IgE (FcepsilonRI) underlies atopic allergic reactions. Curcumin can block this activation, but the mechanism and the effects of curcumin on IgE-mediated allergic reactions are unknown. OBJECTIVES: We sought to determine the antiallergic activity of curcumin in vivo and its mechanism of action in mast cells. METHODS: The antiallergic activity of curcumin was evaluated in mast cell cultures and the passive cutaneous anaphylaxis model. The effects of curcumin on mast cell signaling events were examined by using immunoblotting, immunoprecipitation, RT-PCR, and other molecular biologic approaches. RESULTS: Curcumin inhibited antigen-mediated activation of mast cells and passive cutaneous anaphylaxis in mice. Suppression of degranulation and secretion of TNF-alpha and IL-4 was apparent at concentrations as low as 3 micromol/L curcumin in activated mast cells. Similar concentrations of curcumin suppressed Syk-dependent phosphorylations of the adaptor proteins linker of activated T cells and Grb2-associated binder 2, which are critical for mast cell activation. Although curcumin did not inhibit the phosphorylation of Syk itself, it directly inhibited Syk kinase activity in vitro. Further downstream, activating phosphorylations of Akt and the mitogen-activated protein kinases p38, p44/42 (extracellular signal-regulated kinase 1/2), and c-Jun N-terminal kinase, which are critical for the production of inflammatory cytokines, were also inhibited. CONCLUSIONS: Curcumin inhibits Syk kinase-dependent signaling events in mast cells and might thus contribute to its antiallergic activity. Therefore curcumin might be useful for the treatment of mast cell-related immediate and delayed allergic diseases.

Polygoni cuspidati radix inhibits the activation of Syk kinase in mast cells for antiallergic activity.

The antiallergic activity of Polygoni cuspidati radix (PR) and the mechanism of action by which it functions were investigated in this study. The extract of PR exhibited potent inhibitory activity in mast cells; its IC50 values were 62 +/- 2.1 microg/ml for RBL-2H3 mast cells and 46 +/- 3.2 microg/m for bone marrow-derived mast cells by antigen stimulation, and it also suppressed the expression of tumor necrosis factor-alpha and interleukin-4 in RBL-2H3 cells. According to the in vivo animal allergy model, it inhibited a local allergic reaction, passive cutaneous anaphylaxis, in a dose-dependent manner. With regard to its mechanism of action, PR inhibited the activating phosphorylation of Syk, a key signaling protein for the activation of mast cells. It also suppressed Akt and the mitogen-activated protein kinases ERK1/2, p38, and JNK, which are critical for the production of various inflammatory cytokines in mast cells. The results of the study indicate that the antiallergic activity of PR is mediated through the inhibition of histamine release and allergic cytokine production by the inhibition of Syk activating phosphorylation in mast cells.

Meliae cortex extract exhibits anti-allergic activity through the inhibition of Syk kinase in mast cells.

The anti-allergic action of various Oriental medicinal herbs was investigated using in vitro and in vivo experimental models. Of these extracts, the ethanol extract of Meliae cortex (MC) exhibited the most potent activity in mast cells; its IC(50) values were 29+/-1.5 microg/ml for antigen stimulation and 57+/-3.4 microg/ml for thapsigargin stimulation. It inhibited compound-48/80-induced systemic anaphylaxis by 52.9% at a dose of 300 mg/kg in mice; it also inhibited the expression of the proinflammatory mediator TNF-alpha. With regard to its mechanism of action, MC suppressed the activating phosphorylation of Syk, a key enzyme in mast-cell signaling processes and that of Akt in a dose-dependent manner. It also inhibited the MAP kinase ERK1/2, which is critical for the production of inflammatory cytokines in mast cells, as indicated by the suppression of the activating phosphorylation of ERK1/2. Taken together, these results suggest that the anti-allergic activity of MC may be due to the inhibition of histamine secretion and cytokine expression through the Syk inhibition in mast cells.

Phospholipase D2 acts as an essential adaptor protein in the activation of Syk in antigen-stimulated mast cells.

Mast cells are responsible for IgE-mediated allergic reactions. Phospholipase D1 (PLD1) and PLD2 regulate mast cell activation, but the mechanisms remain unclear. Here we show that PLD2 associates with and promotes activation of Syk, a key enzyme in mast cell activation. Antigen stimulation resulted in increased association and colocalization of Syk with PLD2 on the plasma membrane as indicated by coimmunoprecipitation and confocal microscopy. This association was dependent on tyrosine phosphorylation of Syk but not on PLD2 activity. In vitro, PLD2 interacted via its Phox homology (PX) domain with recombinant Syk to induce phosphorylation and activation of Syk. Furthermore, overexpression of PLD2 or catalytically inactive PLD2K758R enhanced antigen-induced phosphorylations of Syk and its downstream targets, the adaptor proteins LAT and SLP-76, while expression of a PLD2 siRNA blocked these phosphorylations. Apparently, the interaction of PLD2 with Syk is an early critical event in the activation of mast cells.

Inhibitory effects of a fermented ginseng extract, BST204, on the expression of inducible nitric oxide synthase and nitric oxide production in lipopolysaccharide-activated murine macrophages.

In this study, the effects of BST204, a fermented ginseng extract, on the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production are looked into. Crude ginseng extract was incubated with ginsenoside-beta-glucosidase to prepare BST204. BST204, unlike lipopolysaccharide (LPS) and crude ginseng extract, did not affect the level of iNOS protein and NO production in unstimulated RAW 264.7 cells. However, it suppressed the level of iNOS protein and NO production in LPS-stimulated RAW 264.7 cells but did not manifest the same effect on the iNOS mRNA level. An investigation of the activating phosphorylation of p70 S6 kinase and 4E-BP1, which are important for translation, was conducted to investigate the suppressive mechanism of iNOS protein. LPS increased the phosphorylation of p70 S6 kinase, but not 4E-BP1, in a time-dependent manner, and BST204 inhibited it in a dose-dependent manner. The expression of iNOS protein, however, was partially suppressed by rapamycin, an upstream inhibitor of p70 S6 kinase. Therefore, this paper suggests that the suppression of iNOS protein by BST204 was partially correlated with the inhibition of p70 S6 kinase activation.

Effect of a fermented ginseng extract, BST204, on the expression of cyclooxygenase-2 in murine macrophages.

This paper investigates how BST204, a fermented ginseng extract, affects the expression and mechanism of cyclooxygenase-2 (COX-2). BST204 was prepared by incubating crude ginseng extract with ginsenoside-beta-glucosidase. Unexpectedly, BST204 had no effect on the level of COX-2 protein in unstimulated RAW 264.7 cells, and it suppressed the level of COX-2 protein and PGE(2) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. It did not show any suppressive effect, though, on the COX-2 mRNA level. To investigate the suppressive mechanism of COX-2 protein, the activating phosphorylation of p70 S6 kinase and 4E-BP1, which are important for translation, were measured. The phosphorylation of p70 S6 kinase, not 4E-BP1, was increased by LPS in a time-dependent manner, and was inhibited by BST204 in a dose-dependent manner. The expression of COX-2 protein, however, was partially suppressed by rapamycin, an upstream inhibitor of p70 S6 kinase. Therefore, this paper suggests that the suppression of COX-2 protein by BST204 was partially correlated with the inhibition of p70 S6 kinase activation.